Stability assessment of Polatuzumab vedotin and Brentuximab vedotin using different analytical techniques.

Antibody-drug conjugates (ADC) are considered to be fast-growing innovative biopharmaceuticals. The science used for conjugating potent cytotoxic payload to the targeted monoclonal antibody through a chemical linker has played a great value in the area of oncology treatment. In this study; Polatuzumab vedotin (POLA) and Brentuximab vedotin (SGN-35) were subjected to various stress conditions enclosing different pH, thermal stress, agitation, and successive cycles of freeze and thaw in order to produce potential degradation by-products and guarantee the appropriateness of the applied testing protocol. Different analytical techniques were established and validated to be used in the quantitation of the degraded products from different perspectives. The formation of ADC aggregates and fragments was monitored using SE-HPLC as well as dynamic light scattering (DLS). The drug antibody ratio (DAR) and ADC conjugation profile were determined using hydrophobic interaction chromatography (HIC-HPLC). In addition to performing a statistical interpretation of HIC-HPLC results by principal component analysis (PCA) to explicate the obtained data. Also, the quantity of the unconjugated toxic drug was quantified using RP-HPLC. Testing the binding activity of ADC to their target receptor ADC was conducted using ELISA. Results presented that used assay protocol had worked as a complementary design for characterization and stability assessment of the used ADC. Variances in the stability profile of both products were observed which could be attributed to the usage of different formulation buffers. This highlighted the importance of using multiple techniques for the assessment of the quality attributes of such sophisticated products. The analytical assay protocol should be used for the evaluation of the quality and stability of several ADC.

Nanoformulated Recombinant Human Myelin Basic Protein and Rituximab Modulate Neuronal Perturbations in Experimental Autoimmune Encephalomyelitis in Mice.

Introduction: Rituximab (RTX) and recombinant human myelin basic protein (rhMBP) were proven to be effective in ameliorating the symptoms of multiple sclerosis (MS). In this study, a nanoformulation containing rhMBP with RTX on its surface (Nano-rhMBP-RTX) was prepared and investigated in comparison with other treatment groups to determine its potential neuro-protective effects on C57BL/6 mice after inducing experimental autoimmune encephalomyelitis (EAE). Methods: EAE was induced in the corresponding mice by injecting 100 µL of an emulsion containing complete Freund's adjuvant (CFA) and myelin oligodendrocyte glycoprotein (MOG). The subjects were weighed, scored and subjected to behavioural tests. After reaching a clinical score of 3, various treatments were given to corresponding EAE-induced and non-induced groups including rhMBP, RTX, empty nanoparticle prepared by poly (lactide-co-glycolide) (PLGA) or the prepared nanoformulation (Nano-rhMBP-RTX). At the end of the study, biochemical parameters were also determined as interferon-γ (IFN-γ), myeloperoxidase (MPO), interleukin-10 (IL-10), interleukin-4 (IL-4), tumor necrosis factor alpha (TNF-α), nuclear factor kappa B (NF-kB), brain derived neurotrophic factor (BDNF), 2', 3' cyclic nucleotide 3' phosphodiesterase (CNP) and transforming growth factor beta (TGF-ß) along with some histopathological analyses. Results: The results of the Nano-rhMBP-RTX group showed promising outcomes in terms of reducing the clinical scores, improving the behavioural responses associated with improved histopathological findings. Elevation in the levels of IL-4, CNP and TGF-ß was also noticed along with marked decline in the levels of NF-kB and TNF-α. Conclusion: Nano-rhMBP-RTX treated group ameliorated the adverse effects induced in the EAE model. The effectiveness of this formulation was demonstrated by the normalization of EAE-induced behavioral changes and aberrant levels of specific biochemical markers as well as reduced damage of hippocampal tissues and retaining higher levels of myelination.

Green and Cost-Effective Extraction Techniques of Quercetin from Mixture of Nutraceuticals with Yield Analysis via Spectrophotometry and High-Performance Liquid Chromatography Methods.

BACKGROUND: Extraction is the leading critical stage in the analysis of nutraceuticals. Ginkgo biloba (GB) has gained interest because of its therapeutic usages. OBJECTIVES: The aim was to develop four cost-effective extraction techniques for the extraction of quercetin from GB in a sachet containing a mixture of nutraceuticals. These techniques are solid-phase extraction (SPE), liquid-liquid extraction, inverted dispersive liquid-liquid microextraction, and the QuEChERS (quick, easy, cheap, effective, rugged, and safe) method. METHOD: Direct spectrophotometry was used to monitor the recovery of the standard quercetin throughout the optimization steps. The HPLC-UV method of analysis was optimized to quantify the yields from the extracts present in the complicated contents of the sachets. The present study was assessed by analytical Eco-Scale assessment (ESA) and the National Environmental Method Index (NEMI) for greenness in comparison with the literature. RESULTS: SPE showed the best cleanup outcomes. ESA and NEMI showed an adequate greenness of the proposed extraction protocol. CONCLUSIONS: Quercetin (marker for GB) extraction from market nutraceutical sachets is considered an exemplar for analysis in the QC of nutraceuticals. Regarding the greenness results, the proposed method of extraction is better even with adequate greenness as the extraction was a one-step process, in comparison with multistep processes of previously published protocols. Accordingly, it is recommended for use in routine extraction and analysis of such nutraceuticals. HIGHLIGHTS: Four extraction protocols have been developed. For GB ternary-mixture sachets, proper recovery was obtained using C18 SPE. The assessment of greenness of the proposed protocol guaranteed the superiority of the presented method. Safer sorbents and chemicals are favored for use in routine extraction of nutraceuticals.

Impedimetric Sensors for Cyclocreatine Phosphate Determination in Plasma Based on Electropolymerized Poly(o-phenylenediamine) Molecularly Imprinted Polymers.

Cyclocreatine and its water-soluble derivative, cyclocreatine phosphate (CCrP), are potent cardioprotective drugs. Based on recent animal studies, CCrP, FDA-awarded Orphan Drug Designation, has a promising role in increasing the success rate of patients undergoing heart transplantation surgery by preserving donor hearts during transportation and improving the recovery of transplanted hearts in recipient patients. In addition, CCrP is under investigation as a promising treatment for creatine transporter deficiency, an X-linked inborn error resulting in a poor quality of life for both the patients and the caregiver. A newly designed molecularly imprinted polymer (MIP) material was fabricated by the anodic electropolymerization of o-phenylenediamine on screen-printed carbon electrodes and was successfully applied as an impedimetric sensor for CCrP determination to dramatically reduce the analysis time during both the clinical trial phases and drug development process. To enhance the overall performance of the proposed sensor, studies were performed to optimize the electropolymerization conditions, incubation time, and pH of the background electrolyte. Scanning electron microscopy, electrochemical impedance spectroscopy, and cyclic voltammetry were used to characterize the behavior of the developed ultrathin MIP membrane. The CCrP-imprinted polymer has a high recognition affinity for the template molecule because of the formation of 3D complementary cavities within the polymer. The developed MIP impedimetric sensor had good linearity, repeatability, reproducibility, and stability within the linear concentration range of 1 × 10-9 to 1 × 10-7 mol/L, with a low limit of detection down to 2.47 × 10-10 mol/L. To verify the applicability of the proposed sensor, it was used to quantify CCrP in spiked plasma samples.

Coupling of Trastuzumab chromatographic profiling with machine learning tools: A complementary approach for biosimilarity and stability assessment.

Biosimilar products present a growing opportunity to improve the global healthcare systems. The amount of accepted variability during the comparative assessments of biosimilar products introduces a significant challenge for both the biosimilar developers and the regulatory authorities. The aim of this study was to explore unsupervised machine learning tools as a mathematical aid for the interpretation and visualization of such comparability under control and stress conditions using data extracted from high throughput analytical techniques. For this purpose, a head-to-head analysis of the physicochemical characteristics of three Trastuzumab (TTZ) approved biosimilars and the originator product (Herceptin®) was performed. The studied quality attributes included the primary structure and identity by peptide mapping (PM) with reversed-phase chromatography-UV detection, size and charge profiles by stability-indicating size exclusion and cation exchange chromatography. Stress conditions involved pH and thermal stress. Principal component analysis (PCA) and two of the widely used cluster analysis tools, namely, K-means and Density-based Spatial Clustering of Applications with Noise (DBSCAN), were explored for clustering and feature representation of varied analytical datasets. It has been shown that the clustering patterns delineated by the used algorithms changed based on the included chromatographic profiles. The applied data analysis tools were found effective in revealing patterns of similarity and variability between i) intact and stressed as well as ii) originator and biosimilar samples.

Facile imprinted polymer for label-free highly selective potentiometric sensing of proteins: case of recombinant human erythropoietin.

In the current study, a molecularly imprinted polymer (MIP)-based potentiometric sensor was fabricated for a label-free determination of recombinant human erythropoietin (rhEPO). The MIP sensor was operated under zero current conditions using tetra-butyl ammonium bromide as a marker ion. A highly ordered rhEPO surface imprinted layer was prepared using 3-aminopropyl triethoxysilane and tetraethoxysilane as a monomer and cross-linker, respectively, under mild reaction conditions. A two-fold increase in the signal output was obtained by polymeric surface minimization (0.5 mm) that allowed more pronounced molecular recognition (imprinting factor = 20.1). The proportion of cross-reactivity was examined using different interfering biomolecules. Results confirmed sensor specificity for both structurally related and unrelated proteins. An ~40% decrease in the response was obtained for rhEPO-ß compared to rhEPO-α. The imprinted polymeric surface was evaluated using scanning electron microscopy and Fourier transform infrared spectroscopy. Under the optimal measurement conditions, a linear range of 10.00-1000.00 ng mL-1 (10-10 - 10-8 M) was obtained. The sensor was employed for the determination of rhEPO in different biopharmaceutical formulations. Results were validated against standard immunoassay. Spiked human serum samples were analyzed and the assay was validated. The presence of non-specific proteins did not significantly affect (~8%) the results of our assay. A concentration-dependent linear response was produced in an identical range with detection limit as low as 6.50 ng mL-1 (2.14 × 10-10 M). The facile fabricated MIP sensor offers a cost-effective, portable, and easy to use alternative for biosimilarity assessment and clinical application.

Preparation and characterization of 96-well microplates coated with molecularly imprinted polymer for determination and biosimilarity assessment of recombinant human erythropoietin.

Synthesis and applications of molecularly imprinted polymers (MIP) are rapidly growing. In this study, a biomimetic MIP was prepared through silanes polymerization on the surface of 96-well microplates using recombinant human erythropoietin-alfa (rhEPO) as a template molecule. The rhEPO was immobilized onto the plate surface using bi-functional cross-linker and a thin imprinted layer following sol-gel procedure was constructed. After template extraction, uniform three-dimensional cavities compatible with the configuration of rhEPO were obtained. The rhEPO-MIP preparation was optimized using 2-level factorial design and response surface design where polymerization time and interactions between the different variable were found to be the most significant factors. Size-exclusion chromatography (SEC) was used to monitor the stability of the rhEPO under the investigated polymerization conditions. Determination of rhEPO using the MIP microplate showed good dynamic response fitting to the 4 PL regression model (0.9962) over a concentration range of 10.00 - 100.00 ng mL-1. Adsorption of rhEPO onto MIP followed the Langmuir isotherm model (r = 0.9957, χ2 =0.02786) with pseudo-second-order kinetics (r = 0.9984). The surface of the rhEPO-MIP was characterized using scanning electron microscopy (SEM) while step-by-step surface modification was tracked using Fourier transform infrared (FTIR) spectroscopy. The rhEPO-MIP was able to distinguish between the rhEPO-alfa template and modified rhEPO molecules; rhEPO-beta, hyperglycosylated and pegylated forms (imprinting factors < 2) and in the commonly used formulation additive human serum albumin (HSA) (R% = 113.96 -95.22%). The rhEPO-MIP was applied to compare the receptor-binding pattern to rhEPO and its biosimilars / structural analogues. The results were cross-validated using the conventional assay protocol (RP-HPLC and ELISA) and an acceptable correlation was observed with RP-HPLC (maximum deviation is 7.78%). This work confirmed the applicability of rhEPO-MIP with its unique binding features for batch release, stability and biosimilarity assessment as well as subsequent evaluation of batch-to-batch consistency during bioproduction of target analytes.

Analytical comparability study of anti-CD20 monoclonal antibodies rituximab and obinutuzumab using a stability-indicating orthogonal testing protocol: Effect of structural optimization and glycoengineering.

Glycoengineering and biosimilarity are the key factors for growing, promising and progressive approaches in monoclonal antibodies development. In this study, the physicochemical stability of anti-CD20 rituximab (RTX); originator and biosimilar was compared to its glycoengineered humanized version; obinutuzumab (OBZ). An orthogonal stability-indicating protocol using a set of validated bioanalytical techniques; size exclusion high performance liquid chromatography (SE-HPLC), reversed phase liquid chromatography (RP-HPLC), quantitative gel electrophoresis by TapeStation, receptor binding assay and dynamic light scattering (DLS) was used to investigate the effect of different stress factors on the pattern and kinetics of degradation. SE-HPLC results supported with spectral purity showed similar degradation extent with a different pattern of degradation between RTX and OBZ. A lower tendency to form degraded fragments and a relatively higher favorability for degradation through aggregate formation has been revealed in case of OBZ. Results were in agreement with those of DLS and receptor binding assay which showed specificity to the intact antibodies in the presence of their degradation products. Furthermore, results were additionally confirmed through denaturing quantitative gel electrophoresis which suggested reducible covalent bonds as the mechanism for aggregates formation. RP-HPLC results showed two oxidized forms via excessive oxidation of RTX and OBZ with nearly the same degradation percent. Comparability data of RTX and OBZ using the applied methodologies showed that although glycoengineering; carried out to enhance the therapeutic and biological activity of OBZ altered the pattern of degradation but did not significantly affect the overall stability. Results showed also consistent stability profile between the biosimilar and its originator RTX products.

Evaluation of the pattern and kinetics of degradation of adalimumab using a stability-indicating orthogonal testing protocol.

Forced degradation studies are crucial for the evaluation of the stability and biosimilarity. Here, adalimumab was subjected to oxidation, pH, temperature, agitation and repeated freeze-thaw in order to generate all possible degradation products. An orthogonal stability-indicating testing protocol comprising SE-HPLC, RP-HPLC, TapeStation gel electrophoresis, dynamic light scattering (DLS), and functional receptor binding assay was developed and validated. The assay protocol was used for the assessment of the pattern and kinetics of aggregation/degradation of adalimumab. SE-HPLC and DLS were used to show the formation of aggregates/fragments of adalimumab under nondenaturing conditions. TapeStation electrophoresis was performed under denaturing conditions to reveal the nature of aggregates. Results of the receptor binding assay agreed to those of SE-HPLC and DLS which indicated that it can be used as an activity-indicating assay for adalimumab. RP-HPLC demonstrated excellent selectivity for adalimumab in the presence of its oxidized forms. The kinetics of degradation was studied in each case and the results showed that it followed the first-order reaction kinetics. Correlation between the results supported the quality assessment of the tested product in industrial and clinical settings. This orthogonal protocol is a useful tool in stability assessment of monoclonal antibodies and a key criterion for the biosimilarity assessment.

Label-Free Potentiometric Ion Flux Immunosensor for Determination of Recombinant Human Myelin Basic Protein: Application to Downstream Purification from Transgenic Milk.

Recombinant human myelin basic protein (rhMBP) produced in the milk of transgenic cows was found exclusively associated with milk caseins. This hindered its direct determination without extensive sample pretreatment. Here, a label-free potentiometric immunosensor was developed and validated for the determination of rhMBP. An ion flux was generated under zero-current based on surface blocking of the polymeric membrane ion-selective electrode by anti-hMBP antibody and tetrabutylammonium bromide as a marker ion. The immunosensor was successfully employed in the quantitative determination of hMBP in the range of 0.10-20.00 µg/mL with a limit of detection of 50.00 ng/mL. The applicability of the passive ion flux immunosensor for determination of target analyte in complex matrices was investigated. Downstream purification of rhMBP from the milk of transgenic cows was achieved using cation exchange chromatography, immobilized metal affinity chromatography, and immunoaffinity chromatography. The specificity of the immunosensor along with matrix effect of milk proteins were demonstrated. Results obtained using the rhMBP immunosensor were further cross-validated using an orthogonal testing protocol assembled of RP-HPLC and SE-HPLC. It should be noted that the proposed ion flux immunosensor provided a feasible and specific tool for monitoring rhMBP concentration/purity, immunogenic activity, and stability. Such approach provides an attractive economic alternative to sophisticated biosensors required for in-process quality control of biopharmaceutical products.

Simultaneous determination of linezolid, meropenem and theophylline in plasma.

The data presented in this article are related to the research article entitled "Voltammeric monitoring of linezolid, meropenem and theophylline in plasma" (A.K. Attia, M.A. Al-Ghobashy, G.M. El-Sayed, S.M. Kamal, accepted in Anal. Biochem. 2018). This article describes a sensitive square wave voltammetric (SWV) method for simultaneous monitoring of linezolid (LIN), meropenem (MERO) and theophylline (THEO) in spiked plasma and in plasma of healthy volunteers.

Determination of voriconazole and co-administered drugs in plasma of pediatric cancer patients using UPLC-MS/MS: A key step towards personalized therapeutics.

Untreated invasive aspergillosis results in high mortality rate in pediatric cancer patients. Voriconazole (VORI), the first line of treatment, requires strict dose monitoring because of its narrow therapeutic index and individual variation in plasma concentration levels. Commonly co-administered drugs; either Esomeprazole (ESO) or Ondansetron (OND) have reported drug-drug interaction with VORI that should adversely alter therapeutic outcomes of the latter. Although VORI, ESO and OND are co-administered to pediatric cancer patients, the combined effect of ESO and OND on the plasma concentration levels of VORI has not been fully explored. In this study, an accurate, reliable and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed and validated for simultaneous determination of VORI, ESO, and OND in ultra-low sample volumes (25 µL) of plasma of pediatric cancer patients. Based on the physicochemical properties of the studied drugs and internal standard, liquid-liquid extraction was successfully adopted with methyl t-butyl ether. Consistent and reproducible recovery of the three drugs and the internal standard were calculated using plasma and matrix matched samples (RE% > 72.97%, RSD < 8.29%). Chromatographic separation was carried out using UPLC with C18 column and a mobile phase of acetonitrile:water:methanol (70:25:5 V/V/V) at 0.3 mL/min. Mass spectrometric determination at positive electrospray ionization in the MRM mode was employed. The analysis was achieved within 4 min over a linear concentration range of 1.00-200.00 ng/mL for the three drugs. The assay validity was assessed as per the Food and Drug Administration guidelines for bioanalytical method validation, and satisfactory results were obtained. The accuracy and precision were within the acceptable limits for the three drugs in both quality control and incurred plasma samples. Matrix effect and process efficiency were investigated in neat solvent, post-extraction matrix, and plasma. Correlation of the plasma concentration levels of the three drugs revealed differences from the reported drug-drug interactions. This confirmed the need for simultaneous determination of VORI and co-administered drugs in order to achieve optimal therapeutic outcomes. To achieve this, analysis results of this study, genetic polymorphisms in CYP2C19 and clinical data will be used to establish one model incorporating all possible factors that might lead to variation in therapeutic outcomes.

Magnetic molecularly imprinted polymer nanoparticles for simultaneous extraction and determination of 6-mercaptopurine and its active metabolite thioguanine in human plasma.

Cytotoxic drugs used in cancer chemotherapy require the continuous monitoring of their plasma concentration levels for dose adjustment purposes. Such condition necessitates the presence of a sensitive technique for accurate extraction and determination of these drugs together with their active metabolites. In this study a novel solid phase extraction technique using magnetic molecularly imprinted nanoparticles (MMI-SPE) is combined with liquid chromatography tandem mass spectrometry (LC-MS/MS) to extract and determine the anti-leukemic agent; 6-mercaptopurine (6-MP) and its active metabolite thioguanine (TG) in human plasma. The magnetic molecularly imprinted nanoparticles (Fe3O4@MIP NPs) were synthesized via precipitation polymerization technique and were characterized using different characterization methods A computational approach was adopted to help in the choice of the monomer used in the fabrication process. The Fe3O4@MIPs NPs possessed a highly improved imprinting efficiency, fast adsorption kinetics following 2nd order kinetics and good adsorption capacity of 1.0 mg/g. The presented MMI-SPE provided the optimum approach in comparison to other reported ones to achieve good extraction recovery and matrix effect of trace levels of 6-MP and TG from plasma. Chromatographic separation was carried out using a validated LC-MS/MS assay and recovery, matrix effect and process efficiency were evaluated. Recovery of 6-MP and TG was in the range of 85.94-103.03%, while, matrix effect showed a mean percentage recovery of 85.94-97.62% and process efficiency of 85.54-96.18%. The proposed extraction technique is simple, effective and can be applicable to the extraction and analysis of other pharmaceutical compounds in complex matrices for therapeutic drug monitoring applications.

Assessment of pectin-coated magnetite nanoparticles in low-energy water desalination applications.

Novel magnetite nanoparticles (NPs) modified with pectin coating were fabricated, characterized, and evaluated as potential draw solute in a forward osmosis (FO) process for water desalination applications. The prepared NPs had a spherical shape with an average diameter of 200 nm and saturation magnetization of 23.13 emu/g. Thermogravimetric analysis (TGA) and FTIR spectra elucidated the successful pectin coating on magnetite surface. The potential use of the fabricated NPs in water desalination was conducted via a newly developed lab-scale FO system. Deionized water, saline water (0.2, 0.5, and 1 g% NaCl solution), and real well water (TDS = 0.9 g%) were used as feed solutions. In all experiments, the water flux gradually decreased along with the extension of experimental time and NaCl rejection rate by the FO membrane was measured to be higher than 95%. Moreover, it was found that the pectin-coated magnetite NPs demonstrated to be able to draw clean water across the FO membrane from well water with a remarkable salt rejection of 97%. Thus, it is believed that the proposed FO system using pectin-coated magnetite NPs as draw solute can be a promising technique for desalination of well waters in an environmental-friendly and energy-saving manner.

Voltammetric monitoring of linezolid, meropenem and theophylline in plasma.

Treatment of healthcare associated Pneumonia (HCAP) caused by Methicillin-resistant Staphylococcus aureus (MRSA) requires therapeutic protocols formed of linezolid (LIN) either alone or in combination with meropenem (MERO) and theophylline (THEO). The inter-individual pharmacokinetic variations require the development of reliable therapeutic drug monitoring (TDM) tools especially in immunocompromised patients. A sensitive square wave voltammetric sensor using multiwalled carbon nanotubes (MWCNTs) modified carbon paste electrode in Britton-Robinson buffer was developed and validated. Experimental parameters such as pH, percentage of MWCNTs, and pre-concentration time were optimized. The sensor was employed at pH 11.0 for the determination of LIN in plasma within a concentration range of 2.5 × 10-8 - 8.0 × 10-6 mol L-1without interference from co-administered medications. On the other hand, simultaneous monitoring of LIN, MERO and THEO in plasma was feasible at pH 3.0 over concentration ranges of 4.0 × 10-7- 9.0 × 10-5, 8.0 × 10-7- 9.0 × 10-5 and 8.0 × 10-7 - 9.0 × 10-5 mol L-1, respectively. The performance of the proposed sensor was validated and the applicability for TDM has been demonstrated in plasma of healthy volunteers.

Computer-aided design of magnetic molecularly imprinted polymer nanoparticles for solid-phase extraction and determination of levetiracetam in human plasma.

Analytical methods should be accurate and specific to measure plasma drug concentration. Nevertheless, current sample preparation techniques suffer from limitations, including matrix interference and intensive sample preparation. In this study, a novel technique was proposed for the synthesis of a molecularly imprinted polymer (MIP) on magnetic Fe3O4 nanoparticles (NPs) with uniform core-shell structure. The Fe3O4@MIPs NPs were then applied to separate and enrich an antiepileptic drug, levetiracetam, from human plasma. A computational approach was developed to screen the functional monomers and polymerization solvents to provide a suitable design for the synthesized MIP. Different analysis techniques and re-binding experiments were performed to characterize the Fe3O4@MIP NPs, as well as to identify optimal conditions for the extraction process. Adsorption isotherms were best fitted to the Langmuir model and adsorption kinetics were modeled with pseudo-second-order kinetics. The Fe3O4@MIP NPs showed reasonable adsorption capacity and improved imprinting efficiency. A validated colorimetric assay was introduced as a comparable method to a validated HPLC assay for the quantitation of levetiracetam in plasma in the range of 10-80 µg mL-1 after extraction. The results from the HPLC and colorimetric assays showed good precision (between 1.08% and 9.87%) and recoveries (between 94% and 106%) using the Fe3O4@MIP NPs. The limit of detection and limit of quantification were estimated to be 2.58 µg mL-1 and 7.81 µg mL-1, respectively for HPLC assay and 2.32 µg mL-1 and 7.02 µg mL-1, respectively for colorimetric assay. It is believed that synthesized Fe3O4@MIP NPs as a sample clean-up technique combined with the proposed assays can be used for determination of levetiracetam in plasma.

Comparative Assessment of the Effect of Hyper-glycosylation on the Pattern and Kinetics of Degradation of Darbepoetin Alfa using a Stability-Indicating Orthogonal Testing Protocol.

Darbepoetin alfa (DA); hyper-glycosylated Erythropoietin alfa (EPO) is an essential treatment of anemia in patients with chronic kidney failure and cancer. In this study, DA and EPO were subjected to physicochemical stress factors that might be encountered during production, transport and storage (pH, temperature, agitation, repeated freeze-thaw and oxidation). An orthogonal stability-indicating assay protocol comprised of SE-HPLC, RP-HPLC, ELISA and SDS-PAGE was developed and validated to investigate the effect of further glycosylation of DA on the pattern and kinetics of degradation. Results showed a relatively higher stability and lower tendency to form high molecular weight aggregates in the case of DA when compared to EPO, under equivalent stress conditions. Dimers and aggregates were formed for both drugs across the whole pH range and following incubation at temperatures higher than 2-8°C or repeated freeze/thaw. The same observation was noted upon agitation of standard samples prepared in the formulation buffers at high speed and upon oxidation with hydrogen peroxide. The agreement between SE-HPLC, supported with spectral purity data and ELISA confirmed the specificity of both techniques for the intact drugs. Results of RP-HPLC and SDS-PAGE indicated that dimerization occurred through disulfide and bi-tyrosine covalent bonds in the case of pH and oxidation, respectively. It was evident that aggregation was significantly suppressed upon increasing the glycan size and under any of the studied stress factors loss of the glycan has not been observed. These observations supported with the slow kinetics of degradation confirmed the superiority of glyco-engineering over chemical pegylation to enhance the stability of EPO. Formation of such potentially immunogenic product-related impurities at all tested stress factors confirmed the need for orthogonal testing protocols to investigate the complex pattern of degradation of such sensitive products.

Coupling of on-column trypsin digestion-peptide mapping and principal component analysis for stability and biosimilarity assessment of recombinant human growth hormone.

Peptide mapping (PM) is a vital technique in biopharmaceutical industry. The fingerprint obtained helps to qualitatively confirm host stability as well as verify primary structure, purity and integrity of the target protein. Yet, in-solution digestion followed by tandem mass spectrometry is not suitable as a routine quality control test. It is time consuming and requires sophisticated, expensive instruments and highly skilled operators. In an attempt to enhance the fuctionality of PM and extract multi-dimentional data about various critical quality attributes and comparability of biosimilars, coupling of PM generated using immobilized trypsin followed by HPLC-UV to principal component analysis (PCA) is proposed. Recombinant human growth hormone (rhGH); was selected as a model biopharmaceutical since it is available in the market from different manufacturers and its PM is a well-established pharmacopoeial test. Samples of different rhGH biosimilars as well as degraded samples: deamidated and oxidized were subjected to trypsin digestion followed by RP-HPLC-UV analysis. PCA of the entire chromatograms of test and reference samples was then conducted. Comparison of the scores of samples and investigation of the loadings plots clearly indicated the applicability of PM-PCA for: i) identity testing, ii) biosimilarity assessment and iii) stability evaluation. Hotelling's T2 and Q statistics were employed at 95% confidence level to measure the variation and to test the conformance of each sample to the PCA model, respectively. Coupling of PM to PCA provided a novel tool to identify peptide fragments responsible for variation between the test and reference samples as well as evaluation of the extent and relative significance of this variability. Transformation of conventional PM that is largely based on subjective visual comparison into an objective statiscally-guided analysis framework should provide a simple and economic tool to help both manufacturers and regulatory authorities in quality and biosimilarity assessment of biopharmaceuticals.

Stability assessment of antibody-drug conjugate Trastuzumab emtansine in comparison to parent monoclonal antibody using orthogonal testing protocol.

Antibody-drug conjugates (ADC) represent an emerging, novel class of biopharmaceuticals. The heterogeneity originating from the sophisticated structure requires orthogonal analytical techniques for quality and stability assessment of ADC to ensure safety and efficacy. In this study, the stability of Trastuzumab (recombinant humanized IgG1 mAb, targeting HER2 receptor) and its ADC with DM1 (anti-tubulin anticancer drug), Trastuzumab emtansine (T-DM1) were studied. SE-HPLC was used to monitor formation of aggregates and/or fragments of the monoclonal antibodies (mAb). Correlation with the results of reducing and non-reducing sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-PAGE) and dynamic light scattering (DLS) were performed to interpret the obtained results. RP-HPLC was used for assessment of the stability of DM1 in ADC while spectrophotometry was employed to determine drug antibody ratio (DAR) . The studied drugs were subjected to several stress conditions including pH, temperature, mechanical agitation and repeated freeze and thaw to generate possible degradation products and ensure suitability of the assay protocol. The degradation pattern and extent were demonstrated under the indicated stress conditions. The correlation between the results of SE-HPLC and those of SDS-PAGE and DLS ensured the validity of the orthogonal assay protocol and indicated aggregates that were not detected using SE-HPLC. Results showed clearly that T-DM1 is relatively less stable than its parent mAb. This was attributed to the presence of the drug-linker part that is attached to the mAb. RP-HPLC showed that the cytotoxic drug moiety is liable for degradation under the studied conditions resulting in alteration of DAR as well as formation of degradation products. This confirmed the need for more robust coupling chemistries for production of safe and effective ADC and highlighted the importance of orthogonal testing protocols for quality assessment. The assay protocol should be applicable for quality and stability assessment of various ADC.

Evaluation of the combined effects of pegylation and glycosylation on the stability of erythropoietin using a stability-indicating SE-HPLC.

Recombinant human erythropoietin (rhEPO) is a commonly used biopharmaceutical for the treatment of anemia-associated disorders. Epogen; glycosylated erythropoietin (G-EPO) has short half-life and poor stability. Pegylated Epogen (Peg-G-EPO) was introduced to the market to overcome these limitations. The combined effects of pegylation and glycosylation on the stability of Peg-G-EPO was studied. Determination of Peg-G-EPO in the presence of its degradation products was achieved using SE-HPLC. The assay was validated according to ICH guidelines over concentration range of 50.00-320.00 µg/mL (r 0.9999). A mobile phase of 50 mM phosphate buffer (pH 6.5) with 300 mM sodium chloride and 20% ethanol was employed. Isocratic elution was carried out at 0.5 mL/min over run time of 30 min. Peg-G-EPO was found stable towards mechanical agitation only at low concentrations while it was stable towards repeated freeze/thaw; regardless of the concentration. Effect of temperature and pH were also investigated and Peg-G-EPO was found stable within narrow ranges. Results indicated formation of small molecular weight and very high molecular weight aggregates that have been filtered-off the column. Although Peg-G-EPO was found relatively more stable than its non-pegylated but glycosylated version, results indicated the need for careful stability-assessment of Peg-G-EPO.